Recombinant alpha-fetoprotein for treating cancers

ABSTRACT

Disclosed is a method of inhibiting a neoplasm in a mammal, the method includes administering to the mammal a therapeutically effective amount of recombinant human alpha-fetoprotein.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of and claims priority from U.S.application Ser. No. 08/758,757, filed Dec. 3, 1996, now U.S. Pat. No.6,534,479 B1; which is a continuation of and claims priority fromUtility application Ser. No. 08/377,311, filed Jan. 24, 1995, nowabandoned.

BACKGROUND OF THE INVENTION

This invention relates to cancer therapeutics and diagnostic methods.

Cancer is one of the most common of all human diseases, resulting inover 500,000 deaths annually in the United States alone. Cancertypically is detected by the physician as an abnormal growth, or tumor,which causes illness by production of biochemically active molecules, bylocal expansion, or by invasion into neighboring or outlying tissuesites. The symptoms of the illness depend upon the specific molecularproduct(s) of the cancer. Thus, each type of cancer has a characteristicdevelopmental history that describes the likely clinical course of theparticular neoplastic process. For example, it is known that breastcancer spreads most frequently to the lungs, liver, bone, and brain.

Early detection and diagnosis of cancer is often necessary for devisingan optimal treatment plan for a patient. By determining the presence ofearly metastatic disease, treatment can often be designed whichincreases the chance for cure, or delay the development of symptoms if acure is not achievable. Radiographic imaging is one such procedurewidely used for the detection and diagnosis of various disease states,including cancer. For example, radioactive tracers or imaging agents areused for imaging studies to detect sites of human disease. Such tracermolecules are designed to concentrate at a target and define the extentof a tumor or other disease state. Isotopes coupled to monoclonalantibodies, for example, are of clinical interest as applied to cancerscreening. Imaging agents are most effective if they show a highspecific localization at the target site, i.e., a hightarget-to-background contrast. The contrast produced by an imagingagent, e.g., a labelled monoclonal antibody, is largely determined byits biodistribution in vivo. Accordingly, to improve the ability todetect abnormalities such as cancer, the development of imaging agentsspecifically targeted to cancerous cells is considered essential.

As for cancer treatment, although current therapies such as surgery,biologic therapies, radiotherapies, and chemotherapies have saved andimproved countless lives, they remain imperfect solutions. Indeed, amajor clinical problem is that many cancers remain unresponsive to thesetherapies. For example, the capacity to cure disseminated cancer isdependent on combination chemotherapy, alone or together with biologictherapy, surgery, and/or radiotherapy. Moreover, many cancer cells havebeen found to develop resistance to many anti-cancer drugs attenuatingtheir therapeutic effectiveness. Accordingly, the search has begun fornew anti-cancer compounds which can interact with oncogene products,gene regulators, and growth factors and their receptors. Researchemploying the tools of molecular biology promises to provide a new arrayof anti-cancer agents.

SUMMARY OF THE INVENTION

In general, the invention features compositions and methods for theprotection, treatment, and diagnosis of neoplasia, in particular,cancer. The invention is based on my discovery that unglycosylatedrecombinant human alpha-fetoprotein made in a prokaryote (e.g., E. coli)is useful for treating and diagnosing mammals with neoplasms, especiallymalignant tumors, such as breast or prostate carcinomas, and othercarcinomas caused by a proliferation of malignant cells which expressreceptors which are recognized by recombinant human alpha-fetoprotein.

In one aspect, the invention features a method of inhibiting a neoplasmin a mammal (e.g., a human patient), involving administering to themammal a therapeutically effective amount of recombinant humanalpha-fetoprotein or an anti-neoplasm fragment or analog thereof.Preferably, the neoplasm is a malignant tumor (e.g., a breast tumor or aprostate tumor); and the recombinant human alpha-fetoprotein is producedin a prokaryotic cell (e.g., E. coli) and is unglycosylated. Inpreferred embodiments, the cells of the neoplasm express a receptorwhich is recognized by the recombinant human alpha-fetoprotein. Such aneoplasm is generally a carcinoma such as an adenocarcinoma or asarcoma. In preferred embodiments, the neoplasm proliferates in responseto a hormone, e.g, estrogen or androgen. Preferably, administration ofrecombinant human alpha-fetoprotein inhibits the proliferation of cellsof the neoplasm or, alternatively, kills cells of the neoplasm in themammal. The method further includes administering to the mammal achemotherapeutic agent.

In another aspect, the invention features a method of protecting amammal from developing a neoplasm, involving administering to the mammala therapeutically effective amount of recombinant humanalpha-fetoprotein. Preferably, the recombinant human alpha-fetoproteinis produced in a prokaryotic cell (e.g., E. coli) and is unglycosylated.

In another aspect, the invention features a hybrid cytotoxin including arecombinant human alpha-fetoprotein (or a fragment or analog thereof)linked to a cytotoxic agent. Examples of such cytotoxic agents include,without limitation, diphtheria toxin, Pseudomonas exotoxin A; ricin andother plant toxins such as abrin, modeccin, volkensin, viscumin; chloreatoxin (produced by Vibrio cholerae bacteria); the so-called “Shiga-like”toxins (produced by E. coli and other enteric bacteria); Salmonellaheat-labile enterotoxin; and E. coli heat-labile enterotoxin. In otherpreferred embodiments, the cytotoxic agent is non-proteinaceous.Examples of such non-proteinaceous cytotoxic agents include, withoutlimitation, anti-cancer agents such as doxorubicin, as well asα-emitting radionuclides such as astatine and β-emitting nuclides suchas yttrium. Preferably, the cytotoxic agent of the hybrid cytotoxin islinked by a peptide bond to the recombinant human alpha-fetoprotein, andthe hybrid toxin is produced by expression of a genetically engineeredhybrid DNA molecule. In other preferred embodiments, the cytotoxic agentof the hybrid cytotoxin is a protein; such a cytotoxic agent ischemically conjugated to the recombinant human alpha-fetoprotein.

In other aspects, the invention features a detectably-labelledrecombinant human alpha-fetoprotein or a detectably-labelled fragment oranalog thereof capable of binding to a human neoplastic cell. Preferablysuch a molecule is labelled with a radionuclide, e.g., technetium-99m,iodine-125, iodine-131, or indium. Other detectable labels include,without limitation, enzymes, fluorophores, or other moieties orcompounds which emit a detectable signal (e.g., radioactivity,fluorescence, color) or emit a detectable signal after exposure of thelabel to its substrate or, alternatively, the detectable signal can bean epitope recognized by an antibody (e.g., an epitope ofalpha-fetoprotein or an epitope which is specifically engineered intothe recombinant alpha-fetoprotein such as the HA or myc epitopes).Preferably, the molecule targets a malignant tumor (e.g. a breast tumor,a prostate tumor, or a carcinoma) which express a receptor which isrecognized by the recombinant alpha-fetoprotein (or fragment or analogthereof). Typically, such recombinant alpha-fetoprotein is produced in aprokaryotic cell (e.g., E. coli) and is unglycosylated.

Detectably-labelled recombinant human alpha-fetoprotein is useful formethods of imaging a neoplastic cell-containing region in a humanpatient in vivo. In general, the method involves: (a) providing adetectably-labelled molecule of recombinant human alpha-fetoprotein (ora fragment or analog thereof); (b) administering the molecule to thepatient; (c) allowing the labelled molecule to bind and allowing unboundmolecule to be cleared from the region; and (d) obtaining an image ofthe neoplastic cell-containing region. Preferably, the region is thebreast or is the prostate. In other preferred embodiments, the region,without limitation, is liver tissue, is lung tissue, is spleen tissue,is pancreatic tissue, is brain tissue, is lymph tissue, or is bonemarrow. Preferably, the image is obtained using dynamic gammascintigraphy.

Detectably-labelled recombinant human alpha-fetoprotein (or fragment oranalog thereof) can also be used in a method for diagnosing a neoplasmin a mammal (e.g., a human patient). Such a method includes: (a)contacting the biological sample with the detectably-labelled moleculeof recombinant human alpha-fetoprotein; and (b) detecting the labelbound to the sample, where the detection of label above backgroundlevels is indicative that the patient has a neoplasm. Preferably, themethod involves a biological sample including cells fixed and sectionedprior to the contacting step, and the label bound to the sample is boundto areas corresponding to the cell membrane of the cells. In preferredembodiments, the biological sample is from the breast or prostate of ahuman patient.

Detectably-labelled recombinant human alpha-fetoprotein (or fragment oranalog thereof) can also be used in a method for detecting a neoplasm amammal in vivo. Such a method includes: (a) administering adiagnostically effective amount of the detectably-labelled molecule ofrecombinant human alpha-fetoprotein; and (c) detecting the presence ofthe detectable label bound to a tissue of the mammal, where an amount oflabel above background levels is indicative of the presence of theneoplasm in the mammal. In preferred embodiments, the method involves ahuman patient suspected of having a breast cancer, and the tissue isbreast tissue. In other preferred embodiments, the method involves ahuman patient suspected of having a prostate cancer, and the tissue isprostate tissue. Preferably, the detectably labelled recombinant humanalpha-fetoprotein is linked to a radionuclide (e.g., technetium-90) andthe detection step is accomplished by radioimaging (e.g., dynamic gammascintigraphy).

In another aspect, the invention features kits for detecting a neoplasmor any cell expressing a receptor which is recognized by recombinanthuman alpha-fetoprotein (or a fragment or analog thereof) in vivo, insitu or in vitro. In general, the kits include a recombinant humanalpha-fetoprotein which is recognized by a neoplasm, and which may bedetectably labeled. If the recombinant human alpha-fetoprotein isunlabelled, a second reagent containing a detectable label (e.g. aradionuclide such as technetium-90, iodine-125, iodine-131, or indium)is preferably included. Where the detectable label is an enzyme, the kitfurther includes a substrate reagent for the enzyme. The kit may alsoinclude a reagent for linking the detectable label to the recombinantalpha-fetoprotein. In another embodiment, the kit for detecting aneoplasm or any unwanted cell expressing a receptor which is recognizedby recombinant human alpha-fetoprotein (or a fragment or analog thereof)includes a reagent containing an antibody which specifically binds therecombinant human alpha-fetoprotein and a reagent including a detectablylabeled recombinant human alpha-fetoprotein that is specifically boundby the anti-alpha-fetoprotein antibody. Preferably, the recombinanthuman alpha-fetoprotein of the kit is produced in a prokaryotic cell (E.coli) and is unglycosylated.

By “neoplasm” is meant any unwanted growth of cells serving nophysiological function. In general, a cell of a neoplasm has beenreleased from its normal cell division control, i.e., a cell whosegrowth is not regulated by the ordinary biochemical and physicalinfluences in the cellular environment. In most cases, a neoplastic cellproliferates to form a clone of cells which are either benign ormalignant. Examples of neoplasms include, without limitation,transformed and immortalized cells, tumors, and carcinomas such asbreast cell carcinomas and prostate carcinomas.

By “therapeutically effective amount” is meant a dose of unglycosylatedrecombinant human alpha-fetoprotein or an anti-neoplasm fragment oranalog thereof capable of inhibiting the proliferation of a neoplasm.

By “diagnostically effective amount” is meant a dose ofdetectably-labelled recombinant human alpha-fetoprotein or adetectably-labelled fragment or analog thereof that can be detectedwithin a targeted region in a mammal (e.g., a human patient).

By “recombinant human alpha-fetoprotein” is meant a polypeptide havingsubstantially the same amino acid sequence as the protein encoded by thehuman alpha-fetoprotein gene as described by Morinaga et al., Proc.Natl. Acad. Sci., USA 80: 4604 (1983). The method of producingrecombinant human alpha-fetoprotein in a prokaryotic cell is describedin U.S. Pat. No. 5,384,250.

The use of recombinant human alpha-fetoprotein for the treatment anddiagnosis of cancer offers a number of advantages. For example, rHuAFPcan be administered directly to a tumor site. Recombinant HuAFP can alsobe chemically defined and synthesized, and prepared in large quantitiesusing the techniques of recombinant DNA. Moreover, unlike conventionalcancer chemotherapies and radiotherapies, recombinant humanalpha-fetoprotein causes minimal side effects such as nausea, vomiting,and neurotoxicity. Accordingly, relatively high doses of rHuAFP can besafely administered.

The diagnostic methods of the invention are advantageous since in thatthey allow for rapid and convenient diagnosis of a neoplasm. Forexample, the use of rHuAFP as a diagnostic agent (e.g., by radioimagingusing scintigraphy) is especially advantageous for real time imaging ofcancer in both pre-surgical or intraoperative localization and stagingof a cancer, e.g., breast cancer, as well as during post-surgicalexaminations. Using such diagnostic procedures permits non-invasivedetermination of the presence, location, or absence of a neoplasm whichis advantageous for monitoring the condition of a patient. Otherfeatures and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof, and from theclaims.

DETAILED DESCRIPTION

The drawings will first be described.

DRAWINGS

FIG. 1 is the nucleotide sequence (SEQ ID NO: 1) and deduced amino acidsequence (SEQ ID NO: 2) of the cDNA encoding human alpha-fetoprotein.

FIG. 2 is the SDS-PAGE analysis of rHuAFP Fragment I (SEQ ID NO: 8)(Lane A, MW marker; Lane B, natural human alpha-fetoprotein (AFP); LaneC, unpurified rHuAFP; Lane D, rHuAFP Fragment I, and Lane E, rHuAFP(amino acids 1-590 of FIG. 1, SEQ ID NO: 2).

FIG. 3 is a bar graph showing the effect of rHuAFP onestrogen-stimulated post-confluent growth of MCF-7 breast cancer cells.

There now follows a detailed description of methods and compounds of theinvention.

Production of Recombinant Human Alpha-fetoprotein of the Invention

As summarized above, the invention provides compositions for therapiesand diagnostic methods for the prevention and treatment of a neoplasm,involving the use of rHuAFP or anti-neoplasm fragments or analogsthereof. Methods for producing such rHuAFP in a prokaryotic cell aredescribed in U.S. Pat. No. 5,384,250. The invention further providessuch rHuAFP (or a fragment or analog thereof) linked to a toxin or adetectable label for use as anti-cancer or diagnostic agents,respectively. Production of such fragments and analogs of rHuAFP, aswell as anti-cancer and diagnostic agents of rHuAFP, will now bediscussed in greater detail.

Fragments and Analogs

The invention includes biologically active fragments of rHuAFP. Abiologically active fragment of rHuAFP is one that possesses at leastone of the following activities: (a) directs a specific interaction witha target cell, e.g., binds to a cell expressing a receptor which isrecognized by rHuAFP (e.g., the membrane of a cancer cell such as anMCF-7); or (b) halts, reduces, or inhibits the growth of a neoplasm(e.g., binds to a cell surface receptor and imparts ananti-proliferative signal). The ability of rHuAFP fragments or analogsto bind to a receptor which is recognized rHuAFP can be tested using anystandard binding assay known in the art.

Accordingly, a rHuAFP fragment, like the full-length rHuAFP molecule,can be used as a targeting agent for a toxin (for therapy) or adetectable compound (for diagnosis). Recombinant HuAFP fragments thateffectively target a toxin or act as detectable compounds to label acell expressing a receptor which is recognized by rHuAFP or a fragmentor analog thereof are useful in the invention.

In general, fragments of rHuAFP are produced according to the techniquesof polypeptide expression and purification described in (U.S. Pat. No.5,384,250. For example, suitable fragments of rHuAFP can be produced bytransformation of a suitable host bacterial cell with part of anHuAFP-encoding cDNA fragment (e.g., the cDNA described above) in asuitable expression vehicle. Alternatively, such fragments can begenerated by standard techniques of PCR and cloned into the expressionvectors (supra). Accordingly, once a fragment of rHuAFP is expressed, itmay be isolated by various chromatographic and/or immunological methodsknown in the art. Lysis and fractionation of rHuAFP-containing cellsprior to affinity chromatography may be performed by standard methods.Once isolated, the recombinant protein can, if desired, be furtherpurified, e.g., by high performance liquid chromatography (see, e.g.,Fisher, Laboratory Techniques In Biochemistry And Molecular Biology,Work and Burdon, eds., Elsevier, 1980).

A rHuAFP fragment may also be expressed as a fusion protein with maltosebinding protein produced in E. coli. Using the maltose binding proteinfusion and purification system (New England Biolabs, Beverly, Mass.),the cloned human cDNA sequence can be inserted downstream and in frameof the gene encoding maltose binding protein (malE), and the malE fusionprotein can then be overexpressed. In the absence of convenientrestriction sites in the human cDNA sequence, PCR can be used tointroduce restriction sites compatible with the vector at the 5′ and 3′end of the cDNA fragment to facilitate insertion of the cDNA fragmentinto the vector.

Following expression of the fusion protein, it can be purified byaffinity chromatography. For example, the fusion protein can be purifiedby virtue of the ability of the maltose binding protein portion of thefusion protein to bind to amylose immobilized on a column.

To facilitate protein purification, the pMalE plasmid contains a factorXa cleavage site upstream of the site into which the cDNA is insertedinto the vector. Thus, the fusion protein purified as described abovecan then be cleaved with factor Xa to separate the maltose bindingprotein from a fragment of the recombinant human cDNA gene product. Thecleavage products can be subjected to further chromatography to purifyrHuAFP from the maltose binding protein. Alternatively, a fragment ofrHuAFP may be expressed as a fusion protein containing a polyhistidinetag can be produced. Such an alpha-fetoprotein fusion protein may thenbe isolated by binding of the polyhistidine tag to an affinity columnhaving a nickel moiety which binds the polyhistidine region with highaffinity. The fusion protein may then be eluted by shifting the pHwithin the affinity column. The rHuAFP can be released from thepolyhistidine sequences present in the resultant fusion protein bycleavage of the fusion protein with specific proteases.

Recombinant HuAFP fragment expression products (e.g., produced by any ofthe prokaryotic systems described in U.S. Pat. No. 5,384,250) may beassayed by immunological procedures, such as Western blot,immunoprecipitation analysis of recombinant cell extracts, orimmunofluorescence (using, e.g., the methods described in Ausubel etal., Current Protocols In Molecular Biology, Greene PublishingAssociates and Wiley Interscience (John Wiley & Sons), New York, 1994).

Once a fragment of rHuAFP is expressed, it is isolated using the methodsdescribed supra. Once isolated, the fragment of rHuAFP can, if desired,be further purified by using the techniques described supra. Fragmentscan also be produced by chemical synthesis (e.g., by the methodsdescribed in Solid Phase Peptide Synthesis, 2nd ed., 1984, The PierceChemical Co., Rockford, Ill.). The ability of a candidate rHuAFPfragment to exhibit a biological activity of alpha-fetoprotein isassessed by methods known to those skilled in the art (e.g., thosedescribed herein).

The purified recombinant gene product or fragment thereof can then beused to raise polyclonal or monoclonal antibodies against the humanrecombinant alpha-fetoprotein using well-known methods (see Coligan etal., eds., Current Protocols in Immunology, 1992, Greene PublishingAssociates and Wiley-Interscience). To generate monoclonal antibodies, amouse can be immunized with the recombinant protein, andantibody-secreting B cells isolated and immortalized with anon-secretory myeloma cell fusion partner. Hybridomas are then screenedfor production of recombinant human alpha-fetoprotein (or a fragment oranalog thereof)-specific antibodies and cloned to obtain a homogenouscell population which produces monoclonal antibodies.

As used herein, the term “fragment,” as applied to a rHuAFP polypeptide,is preferably at least 20 contiguous amino acids, preferably at least 50contiguous amino acids, more preferably at least 100 contiguous aminoacids, and most preferably at least 200 to 400 or more contiguous aminoacids in length. Fragments of rHuAFP molecules can be generated bymethods known to those skilled in the art, e.g., proteolytic cleavage orexpression of recombinant peptides, or may result from normal proteinprocessing (e.g., removal of amino acids from nascent polypeptide thatare not required for biological activity).

Recombinant HuAFP fragments of interest include, but are not limited to,Domain I (amino acids 1 (Thr)—197 (Ser), see FIG. 1, SEQ ID NO: 3),Domain II (amino acids 198 (Ser)—389 (Ser), see FIG. 1, SEQ ID NO: 4),Domain III (amino acids 390 (Gln)—590 (Val), see FIG. 1, SEQ ID NO: 5),Domain I+II (amino acids 1 (Thr)—389 (Ser), see FIG. 1, SEQ ID NO: 6),Domain II+III (amino acids 198 (Ser)—590 (Val), see FIG. 1, SEQ ID NO:7), and rHuAFP Fragment I (amino acids 266 (Met)—590 (Val), see FIG. 1,SEQ ID NO: 8). Activity of a fragment is evaluated experimentally usingconventional techniques and assays, e.g., the assays described herein.

The invention further includes analogs of full-length rHuAFP orfragments thereof. Analogs can differ from rHuAFP by amino acid sequencedifferences, or by modifications (e.g., post-translationalmodifications) which do not affect sequence, or by both. Analogs of theinvention will generally exhibit at least 80%, more preferably 85%, andmost preferably 90% or even 99% amino acid identity with all or part ofa rHuAFP amino acid sequence. Modifications (which do not normally alterprimary sequence) include in vivo, or in vitro chemical derivatizationof polypeptides, e.g., acetylation, or carboxylation; such modificationsmay occur during polypeptide synthesis or processing or followingtreatment with isolated modifying enzymes. Analogs can also differ fromthe naturally occurring rHuAFP by alterations in primary sequence, forexample, substitution of one amino acid for another with similarcharacteristics (e.g., valine for glycine, arginine for lysine, etc.) orby one or more non-conservative amino acid substitutions, deletions, orinsertions which do not abolish the polypeptide's biological activity.These include genetic variants, both natural and induced (for example,resulting from random mutagenesis by irradiation or exposure toethanemethylsulfate or by site-specific mutagenesis as described inSambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., ColdSpring Harbor Press, 1989, or Ausubel et al., supra)). Also included arecyclized peptide molecules and analogs which contain residues other thanL-amino acids, e.g., D-amino acids or non-naturally occurring orsynthetic amino acids, e.g., β or γ amino acids, or L-amino acids withnon-natural side chains (see e.g., Noren et al., Science 244:182, 1989).Methods for site-specific incorporation of non-natural amino acids intothe protein backbone of proteins is described, e.g., in Ellman et al.,Science 255:197, 1992. Also included are chemically synthesizedpolypeptides or peptides with modified peptide bonds (e.g., non-peptidebonds as described in U.S. Pat. Nos. 4,897,445 and 5,059,653) ormodified side chains to obtain the desired pharmaceutical properties asdescribed herein. Useful mutants and analogs are identified usingconventional methods, e.g., those described herein.

The cloning, expression, isolation and characterization of exemplaryrHuAFP fragments now follows. These examples are provided to illustrate,not limit, the invention.

Experimental

Materials and Methods

Polymerase Chain Reaction (PCR) rHuAFP Fragments

Plasmid constructs encoding fragments of human alpha-fetoprotein wereprepared using polymerase chain reaction (PCR) techniques known to thoseskilled in the art of molecular biology, using oligonucleotide primersdesigned to amplify specific portions of the human alpha-fetoproteingene (see e.g., PCR Technology, H. A. Erlich, ed., Stockton Press, NewYork, 1989; PCR Protocols: A Guide to Methods and Applications, M. A.Innis, David H. Gelfand, John J. Sninsky, and Thomas J. White, eds.,Academic Press, Inc., New York, 1990, and Ausubel et. al., supra).

The following six rHuAFP fragments were prepared to evaluate theirbiological activity (e.g., according to the methods disclosed herein):

Domain I Amino acids 1 (Thr) - 197 (Ser), (FIG. 1, SEQ ID NO: 3) DomainII Amino acids 198 (Ser) - 389 (Ser), (FIG. 1, SEQ ID NO: 4) Domain IIIAmino acids 390 (Gln) - 590 (Val), (FIG. 1, SEQ ID NO: 5) Domain I + IIAmino acids 1 (Thr) - 389 (Ser), (FIG. 1, SEQ ID NO: 6) Domain II + IIIAmino acids 198 (Ser) - 590 (Val), (FIG. 1, SEQ ID NO: 7) rHuAFPFragment I Amino acids 266 (Met) - 590 (Val), (FIG. 1, SEQ ID NO: 8)

Amino acids sequences were deduced from those shown for humanalpha-fetoprotein (1(Thr)—590 (Val); SEQ ID NO: 2) in FIG. 1. Fragmentsof rHuAFP designated Domain I, Domain II, Domain III, Domain I+II,Domain II+III and rHuAFP Fragment I were synthesized using standard PCRreaction conditions in 100 μL reactions containing 34 μL H₂O, 10 μL 10×reaction buffer, 20 μL 1 mM dNTP, 2 μL DNA template (HuAFP cloned inpI18), appropriate 5′ and 3′ oligonucleotide primers (10 μL 10 pmol/μL5′ primer, 10 μL 10 pmol/μL 3′ primer), 1 μL glycerol, 10 μL DMSO, and 1μL Pfu polymerase (Stratagene, LaJolla, Calif.). Primers used for PCRamplifications were:

DomI25 5′-AAAAAAGGTACCACACTGCATAGAAATGAA-3′ (SEQ ID NO: 9) DomI35′-AAAAAAGGATCCTTAGCTTTCTCTTAATTCTTT-3′ (SEQ ID NO: 10) DomII55-′AAAAAAATCGATATGAGCTTGTTAAATCAACAT-3′ (SEQ ID NO: 11) DomII35′-AAAAAAGGATCCTTAGCTCTCCTGGATGTATTT-3′ (SEQ ID NO: 12) DomIII55′-AAAAAAATCGATATGCAAGCATTGGCAAAGCGA-3′ (SEQ ID NO: 13) DomIII35′-AAAAAAGGATCCTTAAACTCCCAAAGCAGCACG-3′ (SEQ ID NO: 14) 5′rHuAFPFragment I 5′-AAAAAAATCGATATGTCCTACATATGTTCTCAA-3′ (SEQ ID NO: 15)

Accordingly, primer pairs DomI25 and DomI3, DomII5 and DomII3, DomIII5and DomIII3, 5′rHuAFP Fragment I and DomIII3, DomI25 and DomII3, andDomII5 and DomIII3 were used to isolate cDNA sequences of Domain I,Domain II, Domain III, rHuAFP Fragment I, Domain I+II, and DomainII+III, respectively, of rHuAFP. Annealing, extension, and denaturationtemperatures were 50° C., 72° C., and 94° C., respectively, for 30cycles. PCR products were purified according to standard methods.Purified PCR products encoding Domain I and Domain I+II were digestedindividually with KpnI and BamHI and cloned separately intoKpnI/BamHI-treated pTrp4. Purified PCR products encoding Domain II,Domain III, Domain II+III, and rHuAFP Fragment I were digestedindividually with Bsp106I and BamHI and were cloned separately intoBsp106I/BamHI-treated pTrp4. Each plasmid construct was subsequentlytransformed into competent E. coli cells. Since the expression productwill begin with the amino acid sequence encoded by the translation startsignal methionine, it is expected that such signal will be removed, orin any event, not affect the bioactivity of the ultimate expressionproduct.

Results

Expression and Purification

E. coli containing the expression plasmid encoding rHuAFP Fragment I wascultured and purified. FIG. 2 (lane D) shows the SDS-PAGE profile of thepurified rHuAFP Fragment I. N-terminal amino acid sequence analysisshowed that rHuAFP Fragment I possessed the amino acid sequenceSer₂₆₇-Tyr-Ile-Cys-Ser-Gln-Gln-Asp-Thr₂₇₅ (SEQ ID NO: 16) whichcorresponds to the expected N-terminal amino acid sequence of rHuAFPFragment I (see FIG. 1, SEQ ID NO: 8) where the initiating methionine iscleaved intracellularly.

Cytotoxic Agents

A hybrid cytotoxin of rHuAFP is prepared by conjugating a full-lengthrHuAFP or a fragment or analog thereof to any number of known toxicentities using conventional techniques. Such toxins are useful forinhibiting the development of a neoplasm (as described infra). Usefulcytotoxins are preferably significantly cytotoxic only when presentintracellularly and are substantially excluded from any given cell inthe absence of a targeting domain. As described below, peptide toxinsfulfill both of these criteria and are readily incorporated into hybridmolecules. If desired, a mixed cytotoxin (i.e., a cytotoxin composed ofall or part of two or more toxins) can also be used. Several usefultoxins are described in more detail below.

Toxin molecules useful in the method of the invention are preferablytoxins, such as peptide toxins, which are significantly cytotoxic onlywhen present intracellularly. of course, under these circumstances themolecule must be able to enter a cell bearing the targeted receptor.This ability depends on the nature of the molecule and the nature of thecell receptor. For example, cell receptors which naturally allow uptakeof a ligand are likely to provide a means for a molecule which includesa toxin to enter a cell bearing that receptor. As is discussed below,the peptide toxin useful in the methods of the invention is fused to arHuAFP (or fragment or analog thereof) binding domain by producing arecombinant DNA molecule which encodes a hybrid protein molecule.

Many peptide toxins have a generalized eukaryotic receptor bindingdomain; in these instances the toxin must be modified to preventintoxication of non-receptor bearing cells. Any such modifications mustbe made in a manner which preserves the cytotoxic functions of themolecule (see U.S. Department of Health and Human Services, U.S. Ser.No. 401,412). Potentially useful toxins include, but are not limited to:cholera toxin, ricin, 0-Shiga-like toxin (SLT-I, SLT-II, SLT II_(V)), LTtoxin, C3 toxin, Shiga toxin, pertussis toxin, tetanus toxin,Pseudomonas exotoxin, saporin, modeccin, and gelanin.

The cytotoxic portion of some molecules useful in the invention, ifdesired, can be provided by a mixed toxin molecule. A mixed toxinmolecule is a molecule derived from two different polypeptide toxins.Generally, as discussed above, polypeptide toxins have, in addition tothe domain responsible for generalized eukaryotic cell binding, anenzymatically active domain and a translocation domain. The binding andtranslocation domains are required for cell recognition and toxin entryrespectively. The enzymatically active domain is the domain responsiblefor cytotoxic activity once the molecule is inside a cell.

Naturally-occurring proteins which are known to have a translocationdomain include diphtheria toxin, Pseudomonas exotoxin A, and possiblyother peptide toxins. The translocation domains of diphtheria toxin andPseudomonas exotoxin A are well characterized (see, e.g., Hoch et al.,Proc. Natl. Acad. Sci. USA 82:1692, 1985; Colombatti et al., J. Biol.Chem. 261:3030, 1986; and Deleers et al., FEBS Lett. 160:82, 1983), andthe existence and location of such a domain in other molecules may bedetermined by methods such as those employed by Hwang et al. Cell48:129, 1987); and Gray et al. Proc. Natl. Acad. Sci. USA 81:2645,1984).

For example, one useful rHuAFP/mixed toxin hybrid molecule is formed byfusing the enzymatically active A subunit of E. coli Shiga-like toxin(see, e.g., Calderwood et al., Proc. Natl. Acad. Sci. USA 84:4364, 1987)to the translocation domain (amino acid residues 202 through 460) ofdiphtheria toxin, and to rHuAFP. The rHuAFP portion of the three-parthybrid causes the molecule to attach specifically to cells bearingreceptors which is recognized by rHuAFP, and the diphtheria toxintranslocation portion acts to insert the enzymatically active A subunitof the Shiga-like toxin into the targeted cell. The enzymatically activeportion of Shiga-like toxin, like diphtheria toxin, acts on the proteinsynthesis machinery of the cell to prevent protein synthesis, thuskilling the cell.

Functional components of the hybrid cytotoxins of the invention arelinked together via a non-covalent or covalent bond, or both.Non-covalent interactions can be ionic, hydrophobic, or hydrophilic,such as interactions involved in a leucine-zipper or antibody-protein Ginteraction (see, e.g., Derrick et al., Nature 359:752, 1992). Anexample of a covalent linkage is a disulfide bond.

A hybrid cytotoxin is prepared by chemically conjugating rHuAFP (orfragment or analog) to a any number of known toxic entities, e.g., thosedescribed above. Such reactions are carried out by standard techniquesknown to those skilled in the art. A typical way of conjugating aprotein to a protein toxin (including,. e.g., bacterial toxins such asdiphtheria toxin or Pseudomonas exotoxin A, or plant toxins such asricin) is by crosslinking through a disulfide bond (see, e.g., Chang etal., J. Biol. Chem. 252:1515, 1977) or a heterobifunctional molecule(see, e.g., Cawley et al. Cell 22:563, 1980). See also Stevens et al.,U.S. Pat. No. 4,894,227.

Alternatively, the hybrid cytotoxin is prepared by expression of ahybrid DNA engineered to encode both the rHuAFP (or a fragment or analogthereof) and the toxin (or a toxic portion thereof), using technologyavailable to those of ordinary skill in the art of making such hybrids(see, e.g., Murphy, U.S. Pat. No. 4,675,382, and Chadhary et al., Proc.Natl. Acad. Sci. USA 84:4538, 1987). For example, a recombinant fusionprotein of rHuAFP and a cytotoxic agent is made according to methodsknown in the art (see, e.g., Murphy supra and Huston et al., Meth.Enzymol. 203:46, 1991). If the hybrid cytotoxin is produced byexpression of a fused gene, a peptide bond serves as the link betweenthe cytotoxic agent and the targeting ligand. Another method useful forconjugating a protein or polypeptide to a protein toxin employs thepolymer, monomethoxy-polyethylene glycol (mPEG), as described in Maitiet al., Int. J. Cancer Suppl. 3:17, 1988.

If desired, following its synthesis, the hybrid cytotoxin is affinitypurified according to standard methods using antibodies against thetargeting portion of the molecule, e.g., antibodies against humanalpha-fetoprotein. Similarly, antibodies directed against the cytotoxicagent are also useful for purifying the hybrid cytotoxin molecule bystandard immunological techniques. The resulting hybrid cytotoxin isthen formulated for use as an agent against unwanted cells, e.g. cancercells, following procedures standard in the field of pharmacology.

Molecules of the invention can be screened for the ability to decreaseviability of cells bearing the targeted receptor by means of assaysknown in the art, e.g., those methods described herein.

Because hybrid cytotoxins of the invention are potent cytotoxic agentsfor cells bearing the a receptor which is recognized by rHuAFP, rHuAFPis useful in the treatment of diseases involving unwantedalpha-fetoprotein receptor-positive cells, e.g., cancer cells.

Diagnostic Agents

Recombinant rHuAFP or a fragment or analog thereof can be attached to adetectable label to produce an agent useful for detecting and localizinga neoplasm in vivo, in situ, or in vitro. Methods for attaching suchlabels to proteins are known in the art. For example, a detectable labelis attached by chemical conjugation, but where the label is apolypeptide, it could alternatively be attached by genetic engineeringtechniques.

Detectable labels are generally selected from a variety of such labelsknown in the art, but are normally radioisotopes, flurophores, enzymes(e.g., horseradish peroxidase), or other moieties or compounds whichemit a detectable signal (e.g., radioactivity, fluorescence, color) oremit a detectable signal after exposure of the label to its substrate.Various detectable label/substrate pairs (e.g., horseradishperoxidase/diaminobenzidine, avidin/streptavidin, luciferase/luciferin,β-galactosidase/X-gal(5-Bromo-4-Chloro-3-Indoyl-D-Galactopyranoside),and methods for labelling proteins for such detection purposes are knownin the art. The usefulness of such an agent can be assayed, for example,by implanting a tumor cell line, e.g, MCF-7, into a host, e.g., a mouse,and determining whether the agent of the invention detectably labels thetumor produced by such implanted cells, e.g., by radioimaging usingscintigraphy. Such an agent can also be used to assay for the presenceof any unwanted cell bearing an alpha-fetoprotein receptor, e.g., byusing Western blot analysis or histochemical staining of a tissuesample, according to known methods.

Recombinant HuAFP as an Anti-cancer Agent

Anti-cancer agents of the invention (e.g., rHuAFP or a fragment oranalog thereof; or a hybrid cytotoxin of rHuAFP) are useful forinhibiting a neoplasm, e.g., breast or prostate carcinomas. Thoseskilled in the art will understand that any number of methods, both invitro and in vivo, are used to determine the efficacy of anti-canceragents useful in the methods of the invention. For example, thereduction of tumor growth can be monitored in a mouse or rat growing aprostate cancer (e.g., tumor xenografts of LNCaP androgenreceptor-positive human prostate cancer cell line) following theadministration of the test compound. In a working example, a human tumorcell lines (e.g., cell lines such as MCF-7(ATCC HTB 22), T-47D (ATCC HTB133), MDA-MB-231 (ATCC HTB 26), BT-20 (ATCC HTB 19), NIH:OV-CAR-3 (ATCCHTB 161), LnCaP.FGC (ATCC CRL 1740), and Du-145 (ATCC HTB 81) growing inculture is released from monolayer by trypsinization, diluted intosingle-cell suspension and then solidified by centrifugation into apellet which IS subsequently exposed to 15 μl fibrinogen (50 mg/ml) and10 μl thrombin (50 units/ml) for 30 minutes at 37° C. Fibrin clotscontaining tumor are then cut into pieces approximately 1.5 mm indiameter. Each piece of tumor is subsequently implanted under the kidneycapsule of a mouse according to standard methods. If desired, mice canbe immunosuppressed by daily subcutaneous (s.c.) injection of 60 mg/kgcyclosporine A (Sandimmune IV) beginning immediately prior to tumorimplantation according to conventional methods. If necessary, estrogenand androgen supplementation of mice is achieved by standard methods,e.g, implantation of silastic tubing containing estradiol or byinjection of testosterone propionate. Typically, hormone supplementationis commenced on the day of tumor implantation. Generally, administrationof the test molecule is initiated prior to tumor implantation and/orafter tumor implantation. Control animals receive a placebo, e.g., humanserum albumin or diluent, similarly administered as for rHuAFP orrelated molecules. The effect of the test molecule on tumor growth ismonitored according to any standard method. For example, tumor growth ismonitored by weekly measurement of tumor size by laparotomy using adissecting microscope equipped with an ocular micrometer. A moleculeshown experimentally to halt or reduce or inhibit the growth of suchimplanted tumors is considered useful in the invention.

Toxicity of test compounds towards cells bearing receptors that arerecognized by rHuAFP can be tested in vitro according to any standardprotocol. For example, a cultured cancer cell line, e.g., MCF-7estrogen-receptor-positive human breast cancer cell line, is maintainedin plastic tissue culture flasks (Costar) in DMEM with penicillin (100U/ml), streptomycin (100 μg/ml), 5% fetal calf serum, insulin (10ng/ml), L-glutamine (2 mM) and non-essential amino acids (1%). Cells areseeded in 96-well V-bottomed plates (Linbro-Flow Laboratories, McLean,Va.) at a concentration of 1×10⁵ per well in complete medium. Putativetoxins are added to varying concentrations (10⁻¹²M to 10⁻⁶M) and thecultures are incubated for 18 hrs. at 37° C. in a 5% CO₂ atmosphere.Following incubation, the plates are centrifuged for 5 min. at 170×g,and the medium removed and replaced with 100 μl leucine-free medium(MEM, Gibco) containing 8 μCi/ml (³H-leucine; New England Nuclear,Boston, Mass.). After an additional 90 min. at 37° C., the plates arecentrifuged for 5 min. at 170×g, the medium is removed, and the cellsare collected on glass fiber filters using a cell harvester (Skatron,Sterling, Va.). Filters are washed, dried, and counted according tostandard methods. Cells cultured with medium alone serve as the control.A test compound which reduces or halts or inhibits cell growth comparedto untreated control cells, is detected as an indication of toxicity andis considered useful in the invention.

Evaluation of whether a test compound confers protection against thedevelopment of a neoplasm (e.g., breast or prostate cancers) generallyinvolves using an animal known to develop a neoplasm (e.g., thetransgenic mouse described in U.S. Pat. No. 4,736,866). An appropriateanimal is treated with the test compound according to standard methods,and a reduced incidence of neoplasm development, compared to untreatedcontrol animals, is detected as an indication of protection.

As is discussed below, I have discovered that unglycosylated rHuAFPproduced in a prokaryotic expression system is effective in treatingcancer. For example, rHuAFP has been found to be a potent inhibitor ofbreast carcinoma growth in vitro.

The experimental examples described below demonstrate the efficacy ofrHuAFP as an anti-cancer agent. These examples are provided toillustrate, not limit, the invention.

Experimental

Materials and Methods

Culture Media and Tumor Cells

Dulbecco's modified Eagle's medium (DMEM), RPMI 1640, fetal calf serum,glutamine, non-essential amino acids and penicillin-streptomycin mixturewere obtained from GIBCO (BRL). Donor calf serum was obtained fromHyclone, Logan, Utah, and porcine insulin was obtained from Squibb,Inc., Princeton, N.J.

The MCF-7 estrogen-receptor-positive human breast cancer cell line wasobtained from Dr. Alberto C. Baldi, Institute of Experimental Biologyand Medicine, Buenos Aires, Argentina. Stock cultures were maintained inplastic tissue culture flasks (Costar) in DMEM with penicillin (100U/ml), streptomycin (100 μg/ml), 5% fetal calf serum, insulin (10ng/ml), L-glutamine (2 mM) and non-essential amino acids (1%).

Synthesis and Purification of rHuAFP

Recombinant HuAFP was synthesized and purified using the methodsdescribed in U.S. Pat. No. 5,384,250. rHuAFP can be obtained fromImmtek, Inc. (Boston, Mass.).

Estrogen-Stimulated Post-confluent Growth of MCF-7 Cells in Culture.

This assay is based on the finding that MCF-7 cells inestrogen-containing medium grow past confluence and accumulate intofoci; but, in the absence of estrogen, cell proliferation stops afterthe cultures establish cell-cell contact, and no foci are formed (see,e.g., Gierthy et al., Breast Cancer Res. Treat. 12:227, 1988). 1×10⁷MCF-7 breast cancer cells were seeded in 16-mm wells contained in24-well tissue culture plates. Culture medium was phenol red-free DMEMsupplemented with 5% donor calf serum (prescreened for absence ofdetectable estrogens), L-glutamine (2 mM), non-essential amino acids(1×, GIBCO), insulin (10 ng/ml), penicillin-streptomycin (1×, GIBCO) andestradiol diluted to a final concentration of 1.8×10⁻⁹ M. Cultures wererefed at 24 hr and every 4 days thereafter with 2 ml of culture mediumcontaining rHuAFP and human serum albumin to yield a final proteinconcentration of 100 μg/ml per well. Cells reached confluence within 5days, and a substantial number of foci were apparent within 10 days inwells containing estrogen alone. Cells were fixed with buffered formalinand stained with 1% Rhodamine B. The stained foci were quantitated usingan Artek 870 Macro-Micro Automated Colony Counter. Data are presented asmean number of foci per treatment group.

Results

Activity of rHuAFP Against MCF-7 Breast Cancer Cells

The results shown in FIG. 3 demonstrate that rHuAFP inhibitsestrogen-stimulated postconfluent growth of MCF-7 breast cancer cells invitro. Control experiments using human albumin or no protein had noeffect on MCF-7 foci formation. These data indicate that rHuAFP has adirect inhibitory effect on the growth of carcinoma cell cultures.

Therapeutic Administration

As demonstrated above, rHuAFP is effective in inhibiting a neoplasm,e.g., a breast cell carcinoma. Accordingly, compounds of the inventioncan be formulated according to known methods to prepare pharmaceuticallyuseful compositions. Treatment of human patients will be carried outusing a therapeutically effective amount of an anti-cancer agent ofrHuAFP in a physiologically acceptable carrier. Suitable carriers andtheir formulation are described for example in Remington'sPharmaceutical Sciences by E. W. Martin. The amount of the anti-canceragent to be administered varies depending upon the manner ofadministration, the age and body weight of the patient, and with thetype of disease, extensiveness of the disease, and size of the patientsuffering from the disease. Generally amounts will be in the range ofthose used for other agents used in the treatment of cancer, although incertain instances lower amounts will be needed because of the increasedspecificity of the compound. For example, rHuAFP is administeredsystemically, as described below, at a dosage that inhibits malignantcell proliferation, typically in the range of 0.1 ng-10 g/kg bodyweight.

Furthermore, the method of the invention can also employ combinationtherapy in which rHuAFP is administered either simultaneously orsequentially with a chemotherapeutic agent. Typically, achemotherapeutic agent is administered according to standard methods or,alternatively, in a dose which is lower than the standard dose when thechemotherapeutic agent is used by itself. Examples of chemotherapeuticagents include, without limitation, mechlorethamine, cyclophosphamide,ifosfamide, L-sarcolysin, chlorambucil, hexamethylmelamine, thiotepa,busulfan, carmustine, lomustine, semustine, streptozocin, dacarbazine,methotrexate, fluorouracil, cytarabine, mercaptopurine, thioguanine,pentostatin, vinblastine, vincristine, etoposide, teniposide,actinomycin D, daunomycin, doxorubicin, bleomycin, plicamycin,mitomycin, cisplatin, mitoxantrone, hydroxyurea, procarbozine, mitotane,aminoglutethimide, prednisone, hydroxyprogesterone, diethylstilbestrol,tamoxifen, flutamide, or leuprolide.

Treatment is started generally with the diagnosis or suspicion of aneoplasm and is generally repeated on a daily basis. Protection from thedevelopment of neoplasm is also achieved by administration of rHuAFP ona daily basis. If desired, the efficacy of the treatment or protectionregimens is assessed with the methods of monitoring or diagnosingpatients for cancer.

Furthermore, the compounds of the invention can also be used to treatmammals to destroy any unwanted cells bearing alpha-fetoproteinreceptors associated with a pathological condition. The method(s) of theinvention can also be used to treat non-human mammals, for example,domestic pets, or livestock. As described below, the anti-cancer agentsof the invention can be administered systemically or locally.

Systemic Administration

For use as an anti-cancer agent, the compounds of the invention can beadministered systemically, for example, formulated in apharmaceutically-acceptable buffer such as physiological saline.Preferable routes of administration include, for example, subcutaneous,intravenous, interperitoneally, intramuscular, or intradermal injectionswhich provide continuous, sustained levels of the drug in the patient.In other preferred routes of administration, the compounds of theinvention can be given to a patient by injection of a slow releasepreparation, for example, in a slowly dissociating polymeric orcrystalline form; this sort of sustained administration can follow aninitial delivery of the drug by more conventional routes (for example,those described above). Alternatively, the compounds can be administeredusing an infusion pump, thus allowing a precise degree of control overthe rate of drug release, or through installation of the compounds inthe nasal passages in a similar fashion to that used to promoteabsorption of insulin. As an alternative to nasal transmucosalabsorption, the compounds can be delivered by aerosol deposition of thepowder or solution into the lungs.

Local Administration

The anti-cancer agents of the invention also can be administered locallyto treat cancer. Since the desired action of the agent is generally upona circumscribed mass of tissue, for example a tumor, delivery of thedrug by means which result in high local concentrations in the vicinityof the tumor is especially desirable.

Recombinant HuAFP as a Diagnostic Agent

Recombinant HuAFP (or fragment or analog thereof) linked to a detectablelabel finds diagnostic use in the detection or monitoring or assayingfor the presence of a neoplasm (e.g., breast or prostate cancers).

For example, in vivo studies can be conducted on human patients todetermine the presence of a neoplasm using a detectably labelled rHuAFP(e.g., Tc-99m-labelled rHuAFP). In general, the detectably labelledrHuAFP is administered intravenously and imaging can be performed usingscanners by methods known to those skilled in the art, e.g., byradioimaging using scintigraphy.

In another working example, a neoplasm or any cell bearing a receptorwhich is recognized by rHuAFP may be detected in a tissue sample, e.g.,a biopsy, a bodily fluid, by using rHuAFP (or fragment or analogthereof) linked to a detectable label. After determining that a patientshould be tested for the presence of such cells, a tissue sample, abiopsy, or a sample of bodily fluid, preferably lymph, blood, serum, orurine, is collected from the patient. Accordingly, the subcellularlocation or presence of a receptor which is recognized by rHuAFP isdetermined either in situ or in vitro using fractionated cells by anystandard biochemical or histochemical procedure (see e.g., Ausubel etal., supra; Bancroft and Stevens, Theory and Practice of HistologicalTechniques, Churchill Livingstone, 1982). Appropriate control samplesfor the assay include a tissue sample or a bodily fluid collected fromindividuals who do not have cells bearing alpha-fetoprotein (negativecontrol), or samples which contain a known, predetermined amount ofalpha-fetoprotein receptor (positive control).

The diagnostic assay may be performed in solution or may use a solid(insoluble) support (e.g. polystyrene, nitrocellulose, or beads) or in atissue sample prepared for histological examination, using any standardmethods. For example, to determine whether the patient from whom thetest sample was collected has cells bearing receptors which isrecognized by rHuAFP, the level of binding of the detectably labelledrHuAFP in the test sample is compared to the level of binding in thenegative and/or positive control samples. A level of binding in the testsample greater than the level of binding in the negative control sample,or at least equal to the level of binding in the positive controlsample, indicates that the patient has cells bearing alpha-fetoproteinreceptors.

Materials for performing the diagnostic assays according to the methodsof the invention may be provided as a kit having instructions for use.In general, the kit is composed in part of a rHuAFP (or fragment oranalog thereof). This kit may further include a second reagent, e.g., adetectable label, which is used to label rHuAFP (or a fragment or analogthereof). The kits exemplified above are useful in, for example,detecting the presence of a tumor in a sample of human tissue in vitro,or for in vivo examination purposes.

The experimental examples described below demonstrate the efficacy ofrHuAFP diagnosing a neoplasm. These examples are provided to illustrate,not limit, the invention.

Experimental

Materials and Methods

Animals

MCF-7 human breast cancer cells implanted in the lateral thorax regionof CB-17 SCID mice were grown to a size of 1 cm diameter (approx. 5 gm)under estrogen stimulation according to methods known in the art.

Technetium Labelling

99mTc-recombinant labelled alpha-fetoprotein was prepared from an AFPaliquot mixed with 0.5 ml 0.9% sodium chloride injection solution(Baxter Healthcare Corporation, Deerfield, Ill.). The solution is addedto an UltraTag RBC Reaction Vial (Mallinckrodt Medical Inc., St. Louis,Mo. 63134 Lot No. 0683040), containing stannous chloride dihydrate,sodium citrate dihydrate, and dextrose anhydrous, in a lyophilized formstored under argon. The contents of the vial are mixed by gentleswirling, and incubated at room temperature for 5 minutes. At thecompletion of the incubation, 0.8-1.2 Gbq Technetium 99mTc SodiumPertechnetate Injection is added (99mTc Generator Mallincrokt Medical,Inc. St. Louis, Mo.) in a volume of 1-2 ml. The contents of the vial aremixed by gentle swirling and incubated for 15 minutes. Dose aliquotswere assayed at 0, 3, and 6 hours after preparation. Thin-layerchromatography performed on preparations using ITLC-SG (GelmanInstrument Co., Ann Arbor, Mich.) with 0.9% NaCl showed 95-99% of the99Tc was bound to the recombinant alpha-fetoprotein.

Imaging

Experimental animals are sedated with Medafane. A 24 gauge, ¾ inchcatheter (Surflo IV catheter, Terumo Medical) is then secured in alateral tail vein. The animal is then further anesthetized with a slowinfusion of 20-25 mg/kg body weight of pentobarbital intravenously.Anesthesia is maintained for restraint as required with injections of 5mg of additional pentobarbital.

Isotope biodistribution data is collected using a Elscint Dymax 409gamma camera. This data is subsequently analyzed by a computer (SiemansGammasonics Microdelta). Animals are imaged in triples, being placed inthe dorsal recumbent position on a thin polyethylene panel. To eliminatemotion during imaging, the animals are restrained as necessary, on thesepanels by strips of tape over their extremities so as not to restrictrespiration. Dynamic images obtained over 60 minutes are used todetermine the biodistribution of the labeled protein. Typically, twelvesequential, five minute images are obtained with low energy generalpurpose collimation, and 1.5 hardware zoom into the computer matriceshaving 128 by 128 picture elements. Study animals are typically injectedwith 37MBq of Tc-99m labelled protein.

Results

Tracer Biodistribution and Kinetics

Following administration of 37MBq (approx. 4-6 μg Tc-99m recombinanthuman alpha-fetoprotein) in the tail vein, tracer biodistributionkinetics were measured during the initial hour after injection and at 24hours. Tissue uptake kinetics were measured in % injected activity/per100 ROI (Region of Interest) pixels (% IA). During the first hour thereis rapid renal clearance, mild localization in the liver and littleevident activity in other tissues. At 1 hour, tumor uptake was(mean±SEM: 1.9±0.3% IA) and the tumor to heart (T/H) region ratio was0.84±0.23. By 24 hours, tumor uptake was (0.8+/−0.1% IA) and T/A andtumor to background (T/B upper chest) region ratios were 1.43±0.41 and2.66±0.54, respectively. Studies comparing 99mTc-labelled rHuAFP to99mTc-labelled human serum albumin (used as a non-specific proteincontrol) repeated in the same animals showed that T/B image ROI activityration was 2.7 and 5.8 for 99mTc-labelled rHuAFP at 1 and 24 hr,respectively and at 24 hours was 40% greater for 99mTc-labelled rHuAFPcompared to Tc-99m human serum albumin. These results show that rHuAFPcan be labelled with Tc-99m and that this labelled agent has lownon-specific tissue uptake and rapid renal clearance from the blood.Localization in human breast cancer xenografts is initially rapid,increases with time, and is due to specific tumor uptake. These resultsdemonstrate that rHuAFP labelled with Tc-99m is useful as a diagnosticagent for breast carcinoma.

Diagnostic Administration

As discussed above, rHuAFP (or fragment or analog thereof) linked to adetectable label finds diagnostic use in the detection or monitoring orassaying of a neoplasm (e.g., a breast cell carcinoma). Accordingly,patients who present with the classical symptoms of cancer, e.g., breastcancer or prostate cancer, or have a medical history which indicatessusceptibility to such cancer may be tested with the methods of theinvention. Other appropriate patients for such testing include those whohave a family history of breast or prostate carcinomas. Patients who arereceiving drugs or have been exposed to toxins implicated in theinduction of a cancer should also be tested.

The diagnostic methods employing detectably labelled rHuAFP (or afragment or analog thereof) of the invention may be used to detect thepresence of a cancer prior to, or after the onset of, clinical symptomsassociated with the cancer.

The method of the invention facilitates diagnosis of a neoplasm prior toor coincident with the onset of clinical symptoms (e.g., a palpabletumorous mass). For example, the method of the subject invention mayprovide a diagnosis of breast cancer prior to onset of clinicalsymptoms. Furthermore, the method of the invention allows the clinicianto provide an accurate diagnosis of a neoplasm such as breast orprostate cancer.

Diagnostic imaging methods of the invention will be carried out using adiagnostically effective amount of a diagnostic agent of rHuAFP in aphysiologically acceptable carrier. Suitable carriers and theirformulation are described for example in Remington's PharmaceuticalSciences by E. W. Martin. The amount of the diagnostic agent to beadministered varies depending upon the manner of administration, the ageand body weight of the patient, and with the type of disease,extensiveness of the disease, and size of the patient suffering from thedisease. Generally, however, amounts will be in the range of those usedfor other agents used in the diagnosis of cancer, although in certaininstances lower amounts will be needed because of the increasedspecificity of the compound. For example, a detectably labelled rHuAFPis administered intravenously to a patient, as is described above, at adosage that allows imaging of a neoplasm, e.g., by radioimaging usingscintigraphy. Typically, a dosage is in the range of 0.1 ng-10 g/kg bodyweight.

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication or patent application was specificallyand individually indicated to be incorporated by reference.

                   #             SEQUENCE LISTING<160> NUMBER OF SEQ ID NOS: 16 <210> SEQ ID NO 1 <211> LENGTH: 2022<212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 1atattgtgct tccaccactg ccaataacaa aataactagc aaccatgaag tg#ggtggaat     60caattttttt aattttccta ctaaatttta ctgaatccag aacactgcat ag#aaatgaat    120atggaatagc ttccatattg gattcttacc aatgtactgc agagataagt tt#agctgacc    180tggctaccat attttttgcc cagtttgttc aagaagccac ttacaaggaa gt#aagcaaaa    240tggtgaaaga tgcattgact gcaattgaga aacccactgg agatgaacag tc#ttcagggt    300gtttagaaaa ccagctacct gcctttctgg aagaactttg ccatgagaaa ga#aattttgg    360agaagtacgg acattcagac tgctgcagcc aaagtgaaga gggaagacat aa#ctgttttc    420ttgcacacaa aaagcccact gcagcatgga tcccactttt ccaagttcca ga#acctgtca    480caagctgtga agcatatgaa gaagacaggg agacattcat gaacaaattc at#ttatgaga    540tagcaagaag gcatcccttc ctgtatgcac ctacaattct tctttcggct gc#tgggtatg    600agaaaataat tccatcttgc tgcaaagctg aaaatgcagt tgaatgcttc ca#aacaaagg    660cagcaacagt tacaaaagaa ttaagagaaa gcagcttgtt aaatcaacat gc#atgtccag    720taatgaaaaa ttttgggacc cgaactttcc aagccataac tgttactaaa ct#gagtcaga    780agtttaccaa agttaatttt actgaaatcc agaaactagt cctggatgtg gc#ccatgtac    840atgagcactg ttgcagagca gatgtgctgg attgtctgca ggatggggaa aa#aatcatgt    900cctacatatg ttctcaacaa gacactctgt caaacaaaat aacagaatgc tg#caaactga    960ccacgctgga acgtggtcaa tgtataattc atgcagaaaa tgatgaaaaa cc#tgaaggtc   1020tatctccaaa tctaaacagg tttttaggag atagagattt taaccaattt tc#ttcagggg   1080aaaaaaatat cttcttggca agttttgttc atgaatattc aagaagacat cc#tcagcttg   1140ctgtctcagt aattctaaga gttgctaaag gataccagga gttattggag aa#gtgtttcc   1200agactgaaaa ccctcttgaa tgccaagata aaggagaaga agaattacag aa#atacatcc   1260aggagagcca agcattggca aagcgaagct gcggcctctt ccagaaacta gg#agaatatt   1320acttacaaaa tgagtttctc gttgcttaca caaagaaagc cccccagctg ac#ctcgtcgg   1380agctgatggc catcaccaga aaaatggcag ccacagcagc cacttgttgc ca#actcagtg   1440aggacaaact attggcctgt ggcgagggag cggctgacat tattatcgga ca#cttatgta   1500tcagacatga aatgactcca gtaaaccctg gtgttggcca gtgctgcact tc#ttcatatg   1560ccaacaggag gccatgcttc agcagcttgg tggtggatga aacatatgtc cc#tcctgcat   1620tctctgatga caagttcatt ttccataagg atctgtgcca agctcagggt gt#agcgctgc   1680aaaggatgaa gcaagagttt ctcattaacc ttgtgaagca aaagccacaa at#aacagagg   1740aacaacttga ggctctcatt gcagatttct caggcctgtt ggagaaatgc tg#ccaaggcc   1800aggaacagga agtctgcttt gctgaagagg gacaaaaact gatttcaaaa ac#tggtgctg   1860ctttgggagt ttaaattact tcaggggaag agaagacaaa acgagtcttt ca#ttcggtgt   1920gaacttttct ctttaatttt aactgattta acactttttg tgaattaatg at#aaagactt   1980 ttatgtgaga tttccttatc acagaaataa aatatctcca aa    #                   #2022 <210> SEQ ID NO 2 <211> LENGTH: 590<212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 2Thr Leu His Arg Asn Glu Tyr Gly Ile Ala Se #r Ile Leu Asp Ser Tyr 1               5   #                10   #                15Gln Cys Thr Ala Glu Ile Ser Leu Ala Asp Le #u Ala Thr Ile Phe Phe            20       #            25       #            30Ala Gln Phe Val Gln Glu Ala Thr Tyr Lys Gl #u Val Ser Lys Met Val        35           #        40           #        45Lys Asp Ala Leu Thr Ala Ile Glu Lys Pro Th #r Gly Asp Glu Gln Ser    50               #    55               #    60Ser Gly Cys Leu Glu Asn Gln Leu Pro Ala Ph #e Leu Glu Glu Leu Cys65                   #70                   #75                   #80His Glu Lys Glu Ile Leu Glu Lys Tyr Gly Hi #s Ser Asp Cys Cys Ser                85   #                90   #                95Gln Ser Glu Glu Gly Arg His Asn Cys Phe Le #u Ala His Lys Lys Pro            100       #           105       #           110Thr Ala Ala Trp Ile Pro Leu Phe Gln Val Pr #o Glu Pro Val Thr Ser        115           #       120           #       125Cys Glu Ala Tyr Glu Glu Asp Arg Glu Thr Ph #e Met Asn Lys Phe Ile    130               #   135               #   140Tyr Glu Ile Ala Arg Arg His Pro Phe Leu Ty #r Ala Pro Thr Ile Leu145                 1 #50                 1 #55                 1 #60Leu Ser Ala Ala Gly Tyr Glu Lys Ile Ile Pr #o Ser Cys Cys Lys Ala                165   #               170   #               175Glu Asn Ala Val Glu Cys Phe Gln Thr Lys Al #a Ala Thr Val Thr Lys            180       #           185       #           190Glu Leu Arg Glu Ser Ser Leu Leu Asn Gln Hi #s Ala Cys Pro Val Met        195           #       200           #       205Lys Asn Phe Gly Thr Arg Thr Phe Gln Ala Il #e Thr Val Thr Lys Leu    210               #   215               #   220Ser Gln Lys Phe Thr Lys Val Asn Phe Thr Gl #u Ile Gln Lys Leu Val225                 2 #30                 2 #35                 2 #40Leu Asp Val Ala His Val His Glu His Cys Cy #s Arg Ala Asp Val Leu                245   #               250   #               255Asp Cys Leu Gln Asp Gly Glu Lys Ile Met Se #r Tyr Ile Cys Ser Gln            260       #           265       #           270Gln Asp Thr Leu Ser Asn Lys Ile Thr Glu Cy #s Cys Lys Leu Thr Thr        275           #       280           #       285Leu Glu Arg Gly Gln Cys Ile Ile His Ala Gl #u Asn Asp Glu Lys Pro    290               #   295               #   300Glu Gly Leu Ser Pro Asn Leu Asn Arg Phe Le #u Gly Asp Arg Asp Phe305                 3 #10                 3 #15                 3 #20Asn Gln Phe Ser Ser Gly Glu Lys Asn Ile Ph #e Leu Ala Ser Phe Val                325   #               330   #               335His Glu Tyr Ser Arg Arg His Pro Gln Leu Al #a Val Ser Val Ile Leu            340       #           345       #           350Arg Val Ala Lys Gly Tyr Gln Glu Leu Leu Gl #u Lys Cys Phe Gln Thr        355           #       360           #       365Glu Asn Pro Leu Glu Cys Gln Asp Lys Gly Gl #u Glu Glu Leu Gln Lys    370               #   375               #   380Tyr Ile Gln Glu Ser Gln Ala Leu Ala Lys Ar #g Ser Cys Gly Leu Phe385                 3 #90                 3 #95                 4 #00Gln Lys Leu Gly Glu Tyr Tyr Leu Gln Asn Gl #u Phe Leu Val Ala Tyr                405   #               410   #               415Thr Lys Lys Ala Pro Gln Leu Thr Ser Ser Gl #u Leu Met Ala Ile Thr            420       #           425       #           430Arg Lys Met Ala Ala Thr Ala Ala Thr Cys Cy #s Gln Leu Ser Glu Asp        435           #       440           #       445Lys Leu Leu Ala Cys Gly Glu Gly Ala Ala As #p Ile Ile Ile Gly His    450               #   455               #   460Leu Cys Ile Arg His Glu Met Thr Pro Val As #n Pro Gly Val Gly Gln465                 4 #70                 4 #75                 4 #80Cys Cys Thr Ser Ser Tyr Ala Asn Arg Arg Pr #o Cys Phe Ser Ser Leu                485   #               490   #               495Val Val Asp Glu Thr Tyr Val Pro Pro Ala Ph #e Ser Asp Asp Lys Phe            500       #           505       #           510Ile Phe His Lys Asp Leu Cys Gln Ala Gln Gl #y Val Ala Leu Gln Arg        515           #       520           #       525Met Lys Gln Glu Phe Leu Ile Asn Leu Val Ly #s Gln Lys Pro Gln Ile    530               #   535               #   540Thr Glu Glu Gln Leu Glu Ala Leu Ile Ala As #p Phe Ser Gly Leu Leu545                 5 #50                 5 #55                 5 #60Glu Lys Cys Cys Gln Gly Gln Glu Gln Glu Va #l Cys Phe Ala Glu Glu                565   #               570   #               575Gly Gln Lys Leu Ile Ser Lys Thr Gly Ala Al #a Leu Gly Val            580       #           585       #           590<210> SEQ ID NO 3 <211> LENGTH: 197 <212> TYPE: PRT<213> ORGANISM: Homo sapiens <400> SEQUENCE: 3Thr Leu His Arg Asn Glu Tyr Gly Ile Ala Se #r Ile Leu Asp Ser Tyr 1               5   #                10   #                15Gln Cys Thr Ala Glu Ile Ser Leu Ala Asp Le #u Ala Thr Ile Phe Phe            20       #            25       #            30Ala Gln Phe Val Gln Glu Ala Thr Tyr Lys Gl #u Val Ser Lys Met Val        35           #        40           #        45Lys Asp Ala Leu Thr Ala Ile Glu Lys Pro Th #r Gly Asp Glu Gln Ser    50               #    55               #    60Ser Gly Cys Leu Glu Asn Gln Leu Pro Ala Ph #e Leu Glu Glu Leu Cys65                   #70                   #75                   #80His Glu Lys Glu Ile Leu Glu Lys Tyr Gly Hi #s Ser Asp Cys Cys Ser                85   #                90   #                95Gln Ser Glu Glu Gly Arg His Asn Cys Phe Le #u Ala His Lys Lys Pro            100       #           105       #           110Thr Ala Ala Trp Ile Pro Leu Phe Gln Val Pr #o Glu Pro Val Thr Ser        115           #       120           #       125Cys Glu Ala Tyr Glu Glu Asp Arg Glu Thr Ph #e Met Asn Lys Phe Ile    130               #   135               #   140Tyr Glu Ile Ala Arg Arg His Pro Phe Leu Ty #r Ala Pro Thr Ile Leu145                 1 #50                 1 #55                 1 #60Leu Ser Ala Ala Gly Tyr Glu Lys Ile Ile Pr #o Ser Cys Cys Lys Ala                165   #               170   #               175Glu Asn Ala Val Glu Cys Phe Gln Thr Lys Al #a Ala Thr Val Thr Lys            180       #           185       #           190Glu Leu Arg Glu Ser         195 <210> SEQ ID NO 4 <211> LENGTH: 192<212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 4Ser Leu Leu Asn Gln His Ala Cys Pro Val Me #t Lys Asn Phe Gly Thr 1               5   #                10   #                15Arg Thr Phe Gln Ala Ile Thr Val Thr Lys Le #u Ser Gln Lys Phe Thr            20       #            25       #            30Lys Val Asn Phe Thr Glu Ile Gln Lys Leu Va #l Leu Asp Val Ala His        35           #        40           #        45Val His Glu His Cys Cys Arg Ala Asp Val Le #u Asp Cys Leu Gln Asp    50               #    55               #    60Gly Glu Lys Ile Met Ser Tyr Ile Cys Ser Gl #n Gln Asp Thr Leu Ser65                   #70                   #75                   #80Asn Lys Ile Thr Glu Cys Cys Lys Leu Thr Th #r Leu Glu Arg Gly Gln                85   #                90   #                95Cys Ile Ile His Ala Glu Asn Asp Glu Lys Pr #o Glu Gly Leu Ser Pro            100       #           105       #           110Asn Leu Asn Arg Phe Leu Gly Asp Arg Asp Ph #e Asn Gln Phe Ser Ser        115           #       120           #       125Gly Glu Lys Asn Ile Phe Leu Ala Ser Phe Va #l His Glu Tyr Ser Arg    130               #   135               #   140Arg His Pro Gln Leu Ala Val Ser Val Ile Le #u Arg Val Ala Lys Gly145                 1 #50                 1 #55                 1 #60Tyr Gln Glu Leu Leu Glu Lys Cys Phe Gln Th #r Glu Asn Pro Leu Glu                165   #               170   #               175Cys Gln Asp Lys Gly Glu Glu Glu Leu Gln Ly #s Tyr Ile Gln Glu Ser            180       #           185       #           190<210> SEQ ID NO 5 <211> LENGTH: 201 <212> TYPE: PRT<213> ORGANISM: Homo sapiens <400> SEQUENCE: 5Gln Ala Leu Ala Lys Arg Ser Cys Gly Leu Ph #e Gln Lys Leu Gly Glu 1               5   #                10   #                15Tyr Tyr Leu Gln Asn Glu Phe Leu Val Ala Ty #r Thr Lys Lys Ala Pro            20       #            25       #            30Gln Leu Thr Ser Ser Glu Leu Met Ala Ile Th #r Arg Lys Met Ala Ala        35           #        40           #        45Thr Ala Ala Thr Cys Cys Gln Leu Ser Glu As #p Lys Leu Leu Ala Cys    50               #    55               #    60Gly Glu Gly Ala Ala Asp Ile Ile Ile Gly Hi #s Leu Cys Ile Arg His65                   #70                   #75                   #80Glu Met Thr Pro Val Asn Pro Gly Val Gly Gl #n Cys Cys Thr Ser Ser                85   #                90   #                95Tyr Ala Asn Arg Arg Pro Cys Phe Ser Ser Le #u Val Val Asp Glu Thr            100       #           105       #           110Tyr Val Pro Pro Ala Phe Ser Asp Asp Lys Ph #e Ile Phe His Lys Asp        115           #       120           #       125Leu Cys Gln Ala Gln Gly Val Ala Leu Gln Ar #g Met Lys Gln Glu Phe    130               #   135               #   140Leu Ile Asn Leu Val Lys Gln Lys Pro Gln Il #e Thr Glu Glu Gln Leu145                 1 #50                 1 #55                 1 #60Glu Ala Leu Ile Ala Asp Phe Ser Gly Leu Le #u Glu Lys Cys Cys Gln                165   #               170   #               175Gly Gln Glu Gln Glu Val Cys Phe Ala Glu Gl #u Gly Gln Lys Leu Ile            180       #           185       #           190Ser Lys Thr Gly Ala Ala Leu Gly Val         195           #       200<210> SEQ ID NO 6 <211> LENGTH: 389 <212> TYPE: PRT<213> ORGANISM: Homo sapiens <400> SEQUENCE: 6Thr Leu His Arg Asn Glu Tyr Gly Ile Ala Se #r Ile Leu Asp Ser Tyr 1               5   #                10   #                15Gln Cys Thr Ala Glu Ile Ser Leu Ala Asp Le #u Ala Thr Ile Phe Phe            20       #            25       #            30Ala Gln Phe Val Gln Glu Ala Thr Tyr Lys Gl #u Val Ser Lys Met Val        35           #        40           #        45Lys Asp Ala Leu Thr Ala Ile Glu Lys Pro Th #r Gly Asp Glu Gln Ser    50               #    55               #    60Ser Gly Cys Leu Glu Asn Gln Leu Pro Ala Ph #e Leu Glu Glu Leu Cys65                   #70                   #75                   #80His Glu Lys Glu Ile Leu Glu Lys Tyr Gly Hi #s Ser Asp Cys Cys Ser                85   #                90   #                95Gln Ser Glu Glu Gly Arg His Asn Cys Phe Le #u Ala His Lys Lys Pro            100       #           105       #           110Thr Ala Ala Trp Ile Pro Leu Phe Gln Val Pr #o Glu Pro Val Thr Ser        115           #       120           #       125Cys Glu Ala Tyr Glu Glu Asp Arg Glu Thr Ph #e Met Asn Lys Phe Ile    130               #   135               #   140Tyr Glu Ile Ala Arg Arg His Pro Phe Leu Ty #r Ala Pro Thr Ile Leu145                 1 #50                 1 #55                 1 #60Leu Ser Ala Ala Gly Tyr Glu Lys Ile Ile Pr #o Ser Cys Cys Lys Ala                165   #               170   #               175Glu Asn Ala Val Glu Cys Phe Gln Thr Lys Al #a Ala Thr Val Thr Lys            180       #           185       #           190Glu Leu Arg Glu Ser Ser Leu Leu Asn Gln Hi #s Ala Cys Pro Val Met        195           #       200           #       205Lys Asn Phe Gly Thr Arg Thr Phe Gln Ala Il #e Thr Val Thr Lys Leu    210               #   215               #   220Ser Gln Lys Phe Thr Lys Val Asn Phe Thr Gl #u Ile Gln Lys Leu Val225                 2 #30                 2 #35                 2 #40Leu Asp Val Ala His Val His Glu His Cys Cy #s Arg Ala Asp Val Leu                245   #               250   #               255Asp Cys Leu Gln Asp Gly Glu Lys Ile Met Se #r Tyr Ile Cys Ser Gln            260       #           265       #           270Gln Asp Thr Leu Ser Asn Lys Ile Thr Glu Cy #s Cys Lys Leu Thr Thr        275           #       280           #       285Leu Glu Arg Gly Gln Cys Ile Ile His Ala Gl #u Asn Asp Glu Lys Pro    290               #   295               #   300Glu Gly Leu Ser Pro Asn Leu Asn Arg Phe Le #u Gly Asp Arg Asp Phe305                 3 #10                 3 #15                 3 #20Asn Gln Phe Ser Ser Gly Glu Lys Asn Ile Ph #e Leu Ala Ser Phe Val                325   #               330   #               335His Glu Tyr Ser Arg Arg His Pro Gln Leu Al #a Val Ser Val Ile Leu            340       #           345       #           350Arg Val Ala Lys Gly Tyr Gln Glu Leu Leu Gl #u Lys Cys Phe Gln Thr        355           #       360           #       365Glu Asn Pro Leu Glu Cys Gln Asp Lys Gly Gl #u Glu Glu Leu Gln Lys    370               #   375               #   380 Tyr Ile Gln Glu Ser385 <210> SEQ ID NO 7 <211> LENGTH: 393 <212> TYPE: PRT<213> ORGANISM: Homo sapiens <400> SEQUENCE: 7Ser Leu Leu Asn Gln His Ala Cys Pro Val Me #t Lys Asn Phe Gly Thr 1               5   #                10   #                15Arg Thr Phe Gln Ala Ile Thr Val Thr Lys Le #u Ser Gln Lys Phe Thr            20       #            25       #            30Lys Val Asn Phe Thr Glu Ile Gln Lys Leu Va #l Leu Asp Val Ala His        35           #        40           #        45Val His Glu His Cys Cys Arg Ala Asp Val Le #u Asp Cys Leu Gln Asp    50               #    55               #    60Gly Glu Lys Ile Met Ser Tyr Ile Cys Ser Gl #n Gln Asp Thr Leu Ser65                   #70                   #75                   #80Asn Lys Ile Thr Glu Cys Cys Lys Leu Thr Th #r Leu Glu Arg Gly Gln                85   #                90   #                95Cys Ile Ile His Ala Glu Asn Asp Glu Lys Pr #o Glu Gly Leu Ser Pro            100       #           105       #           110Asn Leu Asn Arg Phe Leu Gly Asp Arg Asp Ph #e Asn Gln Phe Ser Ser        115           #       120           #       125Gly Glu Lys Asn Ile Phe Leu Ala Ser Phe Va #l His Glu Tyr Ser Arg    130               #   135               #   140Arg His Pro Gln Leu Ala Val Ser Val Ile Le #u Arg Val Ala Lys Gly145                 1 #50                 1 #55                 1 #60Tyr Gln Glu Leu Leu Glu Lys Cys Phe Gln Th #r Glu Asn Pro Leu Glu                165   #               170   #               175Cys Gln Asp Lys Gly Glu Glu Glu Leu Gln Ly #s Tyr Ile Gln Glu Ser            180       #           185       #           190Gln Ala Leu Ala Lys Arg Ser Cys Gly Leu Ph #e Gln Lys Leu Gly Glu        195           #       200           #       205Tyr Tyr Leu Gln Asn Glu Phe Leu Val Ala Ty #r Thr Lys Lys Ala Pro    210               #   215               #   220Gln Leu Thr Ser Ser Glu Leu Met Ala Ile Th #r Arg Lys Met Ala Ala225                 2 #30                 2 #35                 2 #40Thr Ala Ala Thr Cys Cys Gln Leu Ser Glu As #p Lys Leu Leu Ala Cys                245   #               250   #               255Gly Glu Gly Ala Ala Asp Ile Ile Ile Gly Hi #s Leu Cys Ile Arg His            260       #           265       #           270Glu Met Thr Pro Val Asn Pro Gly Val Gly Gl #n Cys Cys Thr Ser Ser        275           #       280           #       285Tyr Ala Asn Arg Arg Pro Cys Phe Ser Ser Le #u Val Val Asp Glu Thr    290               #   295               #   300Tyr Val Pro Pro Ala Phe Ser Asp Asp Lys Ph #e Ile Phe His Lys Asp305                 3 #10                 3 #15                 3 #20Leu Cys Gln Ala Gln Gly Val Ala Leu Gln Ar #g Met Lys Gln Glu Phe                325   #               330   #               335Leu Ile Asn Leu Val Lys Gln Lys Pro Gln Il #e Thr Glu Glu Gln Leu            340       #           345       #           350Glu Ala Leu Ile Ala Asp Phe Ser Gly Leu Le #u Glu Lys Cys Cys Gln        355           #       360           #       365Gly Gln Glu Gln Glu Val Cys Phe Ala Glu Gl #u Gly Gln Lys Leu Ile    370               #   375               #   380Ser Lys Thr Gly Ala Ala Leu Gly Val 385                 3 #90<210> SEQ ID NO 8 <211> LENGTH: 325 <212> TYPE: PRT<213> ORGANISM: Homo sapiens <400> SEQUENCE: 8Met Ser Tyr Ile Cys Ser Gln Gln Asp Thr Le #u Ser Asn Lys Ile Thr 1               5   #                10   #                15Glu Cys Cys Lys Leu Thr Thr Leu Glu Arg Gl #y Gln Cys Ile Ile His            20       #            25       #            30Ala Glu Asn Asp Glu Lys Pro Glu Gly Leu Se #r Pro Asn Leu Asn Arg        35           #        40           #        45Phe Leu Gly Asp Arg Asp Phe Asn Gln Phe Se #r Ser Gly Glu Lys Asn    50               #    55               #    60Ile Phe Leu Ala Ser Phe Val His Glu Tyr Se #r Arg Arg His Pro Gln65                   #70                   #75                   #80Leu Ala Val Ser Val Ile Leu Arg Val Ala Ly #s Gly Tyr Gln Glu Leu                85   #                90   #                95Leu Glu Lys Cys Phe Gln Thr Glu Asn Pro Le #u Glu Cys Gln Asp Lys            100       #           105       #           110Gly Glu Glu Glu Leu Gln Lys Tyr Ile Gln Gl #u Ser Gln Ala Leu Ala        115           #       120           #       125Lys Arg Ser Cys Gly Leu Phe Gln Lys Leu Gl #y Glu Tyr Tyr Leu Gln    130               #   135               #   140Asn Glu Phe Leu Val Ala Tyr Thr Lys Lys Al #a Pro Gln Leu Thr Ser145                 1 #50                 1 #55                 1 #60Ser Glu Leu Met Ala Ile Thr Arg Lys Met Al #a Ala Thr Ala Ala Thr                165   #               170   #               175Cys Cys Gln Leu Ser Glu Asp Lys Leu Leu Al #a Cys Gly Glu Gly Ala            180       #           185       #           190Ala Asp Ile Ile Ile Gly His Leu Cys Ile Ar #g His Glu Met Thr Pro        195           #       200           #       205Val Asn Pro Gly Val Gly Gln Cys Cys Thr Se #r Ser Tyr Ala Asn Arg    210               #   215               #   220Arg Pro Cys Phe Ser Ser Leu Val Val Asp Gl #u Thr Tyr Val Pro Pro225                 2 #30                 2 #35                 2 #40Ala Phe Ser Asp Asp Lys Phe Ile Phe His Ly #s Asp Leu Cys Gln Ala                245   #               250   #               255Gln Gly Val Ala Leu Gln Arg Met Lys Gln Gl #u Phe Leu Ile Asn Leu            260       #           265       #           270Val Lys Gln Lys Pro Gln Ile Thr Glu Glu Gl #n Leu Glu Ala Leu Ile        275           #       280           #       285Ala Asp Phe Ser Gly Leu Leu Glu Lys Cys Cy #s Gln Gly Gln Glu Gln    290               #   295               #   300Glu Val Cys Phe Ala Glu Glu Gly Gln Lys Le #u Ile Ser Lys Thr Gly305                 3 #10                 3 #15                 3 #20Ala Ala Leu Gly Val                 325 <210> SEQ ID NO 9<211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Homo sapiens<400> SEQUENCE: 9 aaaaaaggta ccacactgca tagaaatgaa         #                   #           30 <210> SEQ ID NO 10 <211> LENGTH: 33<212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 10aaaaaaggat ccttagcttt ctcttaattc ttt        #                  #         33 <210> SEQ ID NO 11 <211> LENGTH: 33 <212> TYPE: DNA<213> ORGANISM: Homo sapiens <400> SEQUENCE: 11aaaaaaatcg atatgagctt gttaaatcaa cat        #                  #         33 <210> SEQ ID NO 12 <211> LENGTH: 33 <212> TYPE: DNA<213> ORGANISM: Homo sapiens <400> SEQUENCE: 12aaaaaaggat ccttagctct cctggatgta ttt        #                  #         33 <210> SEQ ID NO 13 <211> LENGTH: 33 <212> TYPE: DNA<213> ORGANISM: Homo sapiens <400> SEQUENCE: 13aaaaaaatcg atatgcaagc attggcaaag cga        #                  #         33 <210> SEQ ID NO 14 <211> LENGTH: 33 <212> TYPE: DNA<213> ORGANISM: Homo sapiens <400> SEQUENCE: 14aaaaaaggat ccttaaactc ccaaagcagc acg        #                  #         33 <210> SEQ ID NO 15 <211> LENGTH: 33 <212> TYPE: DNA<213> ORGANISM: Homo sapiens <400> SEQUENCE: 15aaaaaaatcg atatgtccta catatgttct caa        #                  #         33 <210> SEQ ID NO 16 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Homo sapiens <400> SEQUENCE: 16Ser Tyr Ile Cys Ser Gln Gln Asp Thr  1               5

What is claimed is:
 1. A method of inhibiting a neoplasm in a mammal,said method comprising administering to said mammal a therapeuticallyeffective amount of a compound consisting essentially of recombinanthuman alpha-fetoprotein (SEQ ID NO:2) or anti-neoplasm fragment thereofselected from the group consisting of Domain I residues 1 to 197 (SEQ IDNO:3). Domain II residues 198 to 389 (SEQ ID NO:4), Domain III residues390 to 590 (SEQ ID NO:5). Domain I+II residues 1 to 389 (SEQ ID NO:6)Domain II+III residues 198 to 590 (SEQ ID NO:7), and rhAFP Fragment Iresidues 266 to 590 (SEQ ID NO:8).
 2. The method of claim 1, whereinsaid mammal is a human patient.
 3. The method of claim 1, wherein saidneoplasm is a malignant tumor.
 4. The method of claim 3, wherein saidmalignant tumor is a breast tumor.
 5. The method of claim 1, whereinsaid recombinant human alpha-fetoprotein or fragment thereof is producedin a prokaryotic cell and is unglycosylated.
 6. The method of claim 5,wherein said prokaryotic cell is E. coli.
 7. The method of claim 1,wherein cells of said neoplasm express a receptor which is bound by saidrecombinant human alpha-fetoprotein or fragment thereof.
 8. The methodof claim 1, wherein said neoplasm proliferates in response to estrogen.9. The method of claim 1, wherein said administering inhibitsproliferation of cells of said neoplasm in said mammal.
 10. The methodof claim 1, further comprising administering to said mammal achemotherapeutic agent in an effective dose which is lower than thestandard dose when said chemotherapeutic agent is used by itself. 11.The method of claim 3, wherein said malignant tumor is a prostate tumor.12. The method of claim 1, wherein said neoplasm is a carcinoma.
 13. Themethod of claim 1, wherein said neoplasm is an adenocarcinoma.
 14. Themethod of claim 1, wherein said neoplasm is a sarcoma.